Bottom Line:
Antigen uptake and presentation of native protein antigen was reduced.In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation.This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

ABSTRACTThe signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor-dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

Mentions:
Typical adherent aggregates of DC with fine membrane projections were visible at the periphery of the clusters on day 10–12 and in the following days both cell clusters and DC increased in number and size. The first passage was performed around days 12–14 using EDTA treatment to leave behind only the strongly adherent cells. After 10–12 wk, cultures consisted of single adherent cells with fine cytoplasmic protrusions, adherent cell clusters and cells growing in suspension, some of which had veiled morphology. Cell growth could be continuously maintained (at least over one year) but was strictly dependent on the presence in the culture medium of GM-CSF and fibroblast supernatant (R1 medium). Growth factor deprivation led to cell growth arrest and cell death (Fig. 1). DNA fragments in apoptotic cells were visualized by TUNEL staining, starting from 24–48 h after deprivation, with a number of viable cells decreasing to less than 10% after 1 wk (data not shown). After 6 mo of continuous growth this long-term bulk culture of spleen DC was named D1. The method is highly reproducible given that additional long-term growth factor– dependent DC lines have been independently generated using the same approach.

Mentions:
Typical adherent aggregates of DC with fine membrane projections were visible at the periphery of the clusters on day 10–12 and in the following days both cell clusters and DC increased in number and size. The first passage was performed around days 12–14 using EDTA treatment to leave behind only the strongly adherent cells. After 10–12 wk, cultures consisted of single adherent cells with fine cytoplasmic protrusions, adherent cell clusters and cells growing in suspension, some of which had veiled morphology. Cell growth could be continuously maintained (at least over one year) but was strictly dependent on the presence in the culture medium of GM-CSF and fibroblast supernatant (R1 medium). Growth factor deprivation led to cell growth arrest and cell death (Fig. 1). DNA fragments in apoptotic cells were visualized by TUNEL staining, starting from 24–48 h after deprivation, with a number of viable cells decreasing to less than 10% after 1 wk (data not shown). After 6 mo of continuous growth this long-term bulk culture of spleen DC was named D1. The method is highly reproducible given that additional long-term growth factor– dependent DC lines have been independently generated using the same approach.

Bottom Line:
Antigen uptake and presentation of native protein antigen was reduced.In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation.This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.

ABSTRACTThe signals controlling the checkpoints of dendritic cells (DC) maturation and the correlation between phenotypical and functional maturational stages were investigated in a defined model system of growth factor-dependent immature mouse DC. Three sequential stages of DC maturation (immature, mature, and apoptotic) were defined and characterized. Immature DC (stage 1) had low expression of costimulatory molecules, highly organized cytoskeleton, focal adhesion plaques, and slow motility; accordingly, they were very efficient in antigen uptake and processing of soluble proteins. Further, at this stage most of major histocompatibility complex class II molecules were within cytoplasmic compartments consistent with a poor allostimulatory capacity. Bacteria or cytokines were very efficient in inducing progression from stage 1 towards stage 2 (mature). Morphological changes were observed by confocal analysis including depolymerization of F-actin and loss of vinculin containing adhesive structures which correlates with acquisition of high motility. Antigen uptake and presentation of native protein antigen was reduced. In contrast, presentation of immunogenic peptides and allostimulatory activity became very efficient and secretion of IL-12 p75 was detectable after antigen presentation. This functional DC maturation ended by apoptotic cell death, and no reversion to the immature phenotype was observed.